Klaus Ferlinz

FAS/CD95/APO-I ACTIVATES THE ACIDIC SPHINGOMYELINASE VIA CASPASES

Fas/CD95/Apo-I has been shown to stimulate a variety of molecules including several members of the caspase family and the acidic sphingomyelinase (Martin and Green 1995; Gulbins et al, 1995). Here, we demonstrate that Fas receptor-triggered activation of the acidic sphingomyelinase, consumption of sphingomyelin, release of ceramide, and subsequent activation of JNK and p38-K are regulated by caspases. Inhibition of caspases by Ac-YVAD-chloromethylketone or transient CrmA transfection prevented stimulation of acidic sphingomyelinase, release of ceramide and activation of JNK and p38-K upon Fas-receptor crosslinking. Likewise, Fas triggered apoptosis was almost completely blocked by Ac-YVAD-chloromethylketone or CrmA mediated inhibition of caspases. The results suggest a new signalling cascade from the Fas receptor via caspases to acidic sphingomyelinase, ceramide and JNK/p38-K.

Acid sphingomyelinase is involved in CEACAM receptor-mediated phagocytosis of Neisseria gonorrhoeae

The interaction with human phagocytes is a hallmark of symptomatic Neisseria gonorrhoeae infections. Gonococcal outer membrane proteins of the Opa family induce the opsonin‐independent uptake of the bacteria that relies on CEACAM receptors and an active signaling machinery of the phagocyte. Here, we show that CEACAM receptor‐mediated phagocytosis of Opa52‐expressing N. gonorrhoeae into human cells results in a rapid activation of the acid sphingomyelinase. Inhibition of this enzyme by imipramine or SR33557 abolishes opsonin‐independent internalization without affecting bacterial adherence. Reconstitution of ceramide, the product of acid sphingomyelinase activity, in imipramine‐ or SR33557‐treated cells restores internalization of the bacteria. Furthermore, we demonstrate that CEACAM receptor‐initiated stimulation of other signalling molecules, in particular Src‐like tyrosine kinases and Jun N‐terminal kinases, requires acid sphingomyelinase. These studies provide evidence for a crucial role of the acid sphingomyelinase for CEACAM receptor‐initiated signalling events and internalization of Opa52‐expressing N. gonorrhoeae into human neutrophils.

Sphingosine but not sphingosine-1-phosphate stimulates suicidal erythrocyte death.

Sphingosine kinase 1 phosphorylates sphingosine, which is converted to ceramide by ceramide synthetase. Ceramide triggers eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. Erythrocytes lack sphingosine phosphate-degrading enzymes and thus store large quantities of sphingosine phosphate. The present study explored the influence of sphingosine and sphingosine phosphate on eryptosis. [Ca2+]i, was estimated from Fluo3 fluorescence, cell volume from forward scatter and PS exposure from annexin V-binding in FACS analysis. Sphingosine (0.1 – 10 µM) but not sphingosine-1- phosphate (0.1 – 10 µM) increased [Ca2+]i, decreased cell volume and increased PS-exposure. The observations disclose sphingosine, but not sphingosine-1-phosphate, as a strong inducer of eryptosis.

FTY720-Induced Suicidal Erythrocyte Death

FTY720 is a potent anti-inflammatory drug known to trigger suicidal death or apoptosis of a variety of nucleated cells. Erythrocytes may similarly undergo suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis include cell membrane scrambling and cell shrinkage, which are triggered by increase in cytosolic Ca2+ concentration and ceramide. The present study explored whether FTY720 stimulates eryptosis. Cell membrane scrambling was determined from annexin V-binding, cell shrinkage from forward scatter in FACS analysis, cytosolic Ca2+ concentration from Fluo3 fluorescence, ceramide formation from fluorescence-labeled antibody binding and hemolysis from the hemoglobin concentration in the supernatant. Within 48 hours exposure to FTY720 (10 µM) significantly increased annexin V-binding, decreased forward scatter and increased cytosolic Ca2+ concentration but did not significantly modify ceramide formation. The effects of FTY720 were significantly blunted in the nominal absence of extracelluar Ca2+. In conclusion, at toxic concentrations, FTY720 stimulates suicidal cell death, an effect at least partially due to stimulation of Ca2+ entry.

Deletion of arginine (608) in acid sphingomyelinase is the prevalent mutation among Niemann-Pick disease type B patients from northern Africa.

There is a high incidence of Niemann-Pick type B disease in the Maghreb region of North Africa, which includes Morocco, Algeria and Tunisia. A hypothesis that there may well be a common, predominant mutant acid sphingomyelinase allele responsible for the type B phenotype in this population has been tested. A deletion of an arginine codon at amino acid residue 608 was found in one patient. The same mutation was also observed in another of our cases. An original screening procedure using 3′end digoxigenin-labeled allele-specific oligonucleotides and chemiluminescent detection was developed and used parallel to the conventional assay with 5′-end radiolabeled oligonucleotides. Of the 15 non-related, non-Jewish North African type B patients studied, 12 were homozygous and two compound heterozygous for this deletion (26 ΔR608 alleles/30 mutant alleles). Among type B patients from other geographic regions (France, UK, Italy, Czechoslovakia), this mutation was observed in only one of the 16 alleles studied. Our results indicate that deletion of arginine 608 in the acid sphingomyelinase gene is the highly prevalent mutation underlying Niemann-Pick type B disease in the population of Maghreb. A varying severity of the clinical and enzymatic expression within the non-neuronopathic phenotype has however been observed in patients homozygous for the mutation.

Phosphatidylinositol-3,5-bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Abstract Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of A-SMase, L-?-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P[2]), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P[2] is 0.53 M as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells. Even at concentrations of up to 50 uM, PtdIns3,5P[2] neither decreased plasma membrane associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases ?-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.

Characterization of sphingomyelinase activity released by thrombin-stimulated platelets

In this study we report that human platelets display neutral (nSMase) and acid sphingomyelinase (aSMase) as well as acid ceramidase (aCerase) activity. Cell activation by thrombin resulted in a marked decrease of intracellular aSMase activity, accompanied by the release of enzyme into the medium. In contrast, thrombin treatment did not affect aCerase activity. Two major protein bands of 73 and 70 kDa were recognized by aSMase antibodies in resting platelet lysates and in the medium of stimulated cells. Phorbol esters together with the calcium ionophore A23187 fully reproduced thrombin action on aSMase release. The secreted enzymatic activity was insensitive to digestion with endoglycosidase H but it was stimulated by Zn2+, although to a limited extent compared to aSMase constitutively released by murine endothelial cells. Taken together, these data suggest that secreted aSMase does not originate from the lysosomal compartment but rather from other platelet vesicles.

Molecular basis of acid sphingomyelinase dificiency in a patient with Niemann-Pick disease type A

Niemann-Pick disease, an autosomal recessive lysosomal storage disorder, is caused by deficiency of acid sphingomyelinase. Sequence analysis of mRNA and genomic DNA of fibroblasts of a type A patient showed a single G1729 to A nucleotide transition. This mutation resulted in a substitution of serine for normal glycine at position 577 of the peptide sequence. Amplification of the genomic DNA region around the mutation and subsequent sequencing yielded exclusively the same base change found at the cDNA level. Expression studies with this abnormal cDNA in COS-1 cells revealed a complete loss of enzymatic activity of the mutated protein. These findings indicate that this mutation is responsible for the clinical disease of the patient.

Acid Sphingomyelinase Regulates Platelet Cell Membrane Scrambling, Secretion, and Thrombus Formation

Objective— Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation. Approach and Results— Collagen-related peptide–induced or thrombin-induced ATP release and P-selectin exposure were significantly blunted in platelets from Asm-deficient mice (Smpd1 −/−) when compared with platelets from wild-type mice (Smpd1 +/+ ). Moreover, phosphatidylserine exposure and thrombin generation were significantly less pronounced in Smpd1 −/− platelets than in Smpd1 +/+ platelets. In contrast, platelet integrin &agr;IIb&bgr;3 activation and aggregation, as well as activation-dependent Ca2+ flux, were not significantly different between Smpd1 −/− and Smpd1 +/+ platelets. In vitro thrombus formation at shear rates of 1700 s−1 and in vivo thrombus formation after FeCl3 injury were significantly blunted in Smpd1 −/− mice while bleeding time was unaffected. Asm-deficient platelets showed significantly reduced activation-dependent ceramide formation, whereas exogenous ceramide rescued diminished platelet secretion and thrombus formation caused by Asm deficiency. Treatment of Smpd1 +/+ platelets with bacterial sphingomyelinase (0.01 U/mL) increased, whereas treatment with functional acid sphingomyelinase-inhibitors, amitriptyline or fluoxetine (5 &mgr;mol/L), blunted activation-dependent platelet degranulation, phosphatidylserine exposure, and thrombus formation. Impaired degranulation and thrombus formation of Smpd1 −/− platelets were again overcome by exogenous bacterial sphingomyelinase. Conclusions— Acid sphingomyelinase is a completely novel element in the regulation of platelet plasma membrane properties, secretion, and thrombus formation.

Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase

Abstract Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of sphingolipid activator proteins (Saps: saposins A-D; GM2 activator) is not essential to mediate this reaction at the water-lipid interface in vivo. A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the sphingolipid activator protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C.